ACETYLATION OF P45/NF E2 BY PCAF AND ITS CONSEQUENCES

造血是多潜能祖细胞获得谱系特异性表型，并最终形成成熟的循环血细胞的过程。控制造血的环境信号最终由一组转录因子解释，这些转录因子调节与谱系特定功能相关的基因的表达。这项提议的长期目标是定义导致造血转录因子的调节和激活的信号事件。本研究旨在研究乙酰基转移酶PCAF对红系/巨核系转录因子NF-E2的调控作用。PCAF发现核因子-E2的造血亚单位P45在高度保守的赖氨酸残基上发生乙酰化。这一观察结果形成了我们假设P45的活性可能受乙酰化调控的基础。其具体目的是：1)确定P45乙酰化的机制后果。P45a在参与DNA结合的CnC结构域被PCAF乙酰化。P45乙酰化对DNA结合的影响将被确定。乙酰化还可以改变P45的构象，从而调节其与其他蛋白质的相互作用。最后，缺失乙酰化位点的突变体P45的转录活性将通过基因互补在P45缺失的红系细胞中进行检测。2)监测巨核细胞和红系分化过程中P45的乙酰化。为了建立p45乙酰化/去乙酰化与p45靶基因在分化细胞中的激活之间的关系，将产生特异性识别乙酰化p45的抗体。包含体内乙酰化位点的多肽将被用来产生乙酰化P45的特异性抗体。P45的乙酰化状态将在白血病细胞系和原代细胞分化过程中进行跟踪。我们将研究诱导分化的化学物质和细胞因子对p45乙酰化的影响。总之，这些研究可能会对乙酰基转移酶对造血转录因子的调控产生重要的影响。由于这些酶为药物干预提供了极好的靶点，这一知识可能有助于设计用于影响血液疾病患者基因表达和细胞分化的药物。

Hematopoiesis is the process in which pluripotential progenitors acquire lineage-specific phenotype and eventually give rise to mature circulating blood cells. Environmental signals that control hematopoiesis are ultimately interpreted by sets of transcription factors which regulate the expression of genes associated with lineage-specific functions. The long-term objectives of this proposal are to define the signaling events leading to the modulation and activation of hematopoietic transcription factors. This proposal aims at characterizing the regulation of the erythroid/megakaryocytic transcription factor NF-E2 by the acetyltransferase PCAF. The hematopoietic subunit of NF-E2, p45, was found to be acetylated by PCAF at a highly conserved lysine residue. This observation forms the basis of our hypothesis that the activity of p45 may be modulated by acetylation. The specific aims are to: 1) Determine the mechanistic consequences of p45 acetylation. p45 a is acetylated by PCAF in the CNC domain which participates in DNA binding. The effects of p45 acetylation on DNA binding will be determined. Acetylation could also change the conformation of p45 thus could modulate its interaction with other proteins. Finally, the transcriptional activity of mutant p45 lacking the acetylation site will be examined by gene complementation in p45-null erythroid cells. 2) Monitor acetylation of p45 during megakaryocytic and erythroid differentiation. To establish the correlation between p45 acetylation/deacetylation and the activation of p45 target genes in differentiating cells, antibodies specifically recognizing acetylated p45 will be generated. A pepetide encompassing the in vivo acetylation site will be used to generate antibodies specific for acetylated p45. The acetylation status of p45 will be followed in both leukemic cell lines and primary cells undergoing differentiation. The effects of both differentiation-inducing chemicals and cytokines on p45 acetylation will be examined. Together, these studies may yield important insights into the regulation of hematopoietic transcription factors by acetyltransferases. Since these enzymes present excellent targets for pharmacological intervention, this knowledge could be useful for the design of drugs used to influence gene expression and cellular differentiation in patients with hematological disorders.